All-trans retinoic acid regulates the expression of the extracellular matrix protein fibulin-1 in the guinea pig sclera and human scleral fibroblasts.

Purpose Fibulin-1 (FBLN1) mRNA is expressed in human sclera and is an important adhesion modulatory protein that can affect cell–matrix interactions and tissue remodeling. Scleral remodeling is influenced by all-trans retinoic acid (RA). Our purpose was to confirm the presence of fibulin-1 protein in guinea pig sclera and investigate the effect of RA on the expression of fibulin-1 in guinea pig sclera in vivo and in cultured human scleral fibroblasts (HSFs). Methods Confocal fluorescence microscopy was used to study fibulin-1 and aggrecan expression and localization in sclera from control guinea pigs and in animals given RA by daily gavage from 4 to 8 days of age. The effects of RA (from 10−9 to 10−5 M) on fibulin-1 expression in HSFs were observed by immunohistochemistry and assayed by real-time PCR and western blot analysis. Results Fibulin-1 protein expression was detected by confocal fluorescence microscopy in guinea pig sclera and in cultured HSFs. Upregulation of fibulin-1 in scleral tissue was observed after feeding with RA. In vitro, the level of Fbln1 mRNA was increased after treatment of HSFs with RA (at concentrations of 10−8 to 10−6 M; p<0.001), with a maximum effect at 10−7 M. Fibulin-1 protein levels were significantly increased after treatment of HSFs with 10−7 M of RA for 24 or 48 h (p<0.05). Conclusions Fibulin-1 protein was expressed in guinea pig sclera and cultured HSFs. Expression was regulated by RA, a molecule known to be involved in the regulation of eye growth. Further studies on the role of fibulin-1 in the regulation of eye growth, including during the development of myopia, are therefore warranted.

regulate ECM metabolism in many systems [8]. It is an important molecular signal in the control of eye size, perhaps acting as a signal for the direction of ocular growth [9,10]. Although the mechanism by which RA regulates ocular size is not clear, it may affect scleral ECM during scleral remodeling [10]. RA can regulate aggrecan expression and skeletal growth and articular cartilage remodeling in skeletal systems [11]. We sought here to determine whether RA affects the expression of aggrecan and fibulin-1 in guinea pig sclera. It has been shown that six RA receptor subtypes are present in cultured human scleral fibroblasts (HSFs) [12], suggesting that RA may interact with these cells. However, whether RA treatment affects the expression of fibulin-1 in HSFs has not been studied.
The purpose of this study was first to examine the localization of fibulin-1 protein in guinea pig sclera and the presence of fibulin-1 protein in cultured HSFs. Second, we examined whether the expression of fibulin-1 in guinea pig sclera (in vivo) was regulated by RA given by daily gavage [13] and in cultured HSFs treated with RA (in vitro).
They were reared in litters with their mother until 9 days of age when scleral tissue was extracted. During rearing, animals were housed in plastic boxes (65×45×30 cm) lined with a bed of paper pellets and with stainless steel wire lids. Lighting was on a 12 h:12 h light-dark cycle and was provided by overhead incandescent lamps evenly diffused through a perspex barrier located 15 cm above the cages. The average light intensity in the cage was 300 lux. To treat the sclera with RA exogenously, during rearing, young guinea pigs were fed 0.5 ml of RA (24 mg/kg, n=8 eyes) or carrier alone (peanut oil [PO] n=4 eyes) from 4 to 8 days of age every day, 1 h after the beginning of the light cycle. Half of each litter was given either RA or PO to ensure a matched-pair design. Feeding was by gavage while the guinea pigs were lightly anesthetized with isoflurane (1.5% in O2). At 8 days of age, 24 h after the day 7 gavage and before the day 8 gavage, eyes were cyclopleged with 1% cyclopentolate. One hour later, refractive error was measured using streak retinoscopy followed by measurement of axial ocular dimensions in animals using A-scan ultrasonography. These measures were the same as previously described [9] and were used to confirm that the RA treatment had affected ocular elongation First, the animals were lightly anesthetized with 1.5% isoflurane in oxygen. Then, the anesthetized animals were restrained to allow alignment of the transducer probe to the center of the pupil along the optic axis. Approximately 10 measures were taken from each eye sequentially, with the probe realigned several times. On day 9, animals were euthanized by intraperitoneal overdose of barbiturate, eyes were removed on ice, and tissue extracted for immunohistochemistry. All procedures were in compliance with the New South Wales Animal Research Act and approved by the Animal Ethics Committee of the University of Newcastle, Newcastle, Australia. Immunohistochemistry on guinea pig sclera: To observe the expression and localization of fibulin-1 in scleral tissue and find the relationship between fibulin-1 and aggrecan, immunohistochemical staining was observed in scleras from animals fed PO or RA. On day 9 (24 h after the last gavage), eyes were removed and eye cups (without the vitreous) were fixed for 30 min at 20 °C in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed samples were washed three times in PBS (0.05 M phosphate buffer, 195 mM NaCl, and 3 mM NaN3, pH 7.4), cryoprotected in PBS plus 30% sucrose, soaked in embedding medium (optimal cutting temperature compound) for 10 min, and freeze-mounted onto sectioning blocks. Vertical sections (10-12 μm thick) were cut from the posterior pole of the eye and thaw mounted onto gelatincoated glass slides. Sections from RA-and PO-treated eyes were placed together in pairs on each slide to ensure equal exposure to reagents. Sections were air dried, ringed with rubber cement to form a well for antibody solutions, and stored at −20 °C until use. Human scleral fibroblast isolation and identification: This part of the study was approved by the Ethics Committee of Sun Yat-sen University, Guangzhou, China, and complied with the tenets of the Declaration of Helsinki for Research Involving Human Tissue. Normal human eyes (n=8) from donors ranging from 10 to 20 years of age were obtained from the Eye Bank of Zhongshan Ophthalmic Center (Sun Yat-sen University) and were used to make cultures of HSFs. Eyes were washed immediately in Hank's balanced salt solution (HBSS; Gibco, Grand Island, NY) with penicillin (200 μg/ml penicillin-streptomycin) (Invitrogen, Carlsbad, CA) and gentamicin sulfate (400 μg/ml; Invitrogen). The retinas and choroids were removed from the sclera. The posterior sclera was trimmed into 1×1-mm 2 pieces, placed in DMEM/F12 (Gibco) with 1× antibiotic/antimycotic (penicillinstreptomycin; Invitrogen), and 10% fetal bovine serum (Gibco), and then incubated at 37 °C in a humidified incubator containing 5% CO2. The growth medium was changed every 3 days. When a heavy primary monolayer was achieved, cells were trypsinized for 2 min at room temperature in 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA) solution in PBS (Gibco). Cells were subcultured at a split ratio of 1:3 in a 25-mm 2 plastic bottle. The third passage of fibroblasts was used for this experiment. The fibroblasts were grown on coverslips in six-well plates (Corning Ltd., Tokyo, Japan) to 70%-80% confluence. The cells were washed with PBS three times, fixed with acetone for 15 min, air dried, and kept frozen at −20 °C until use. The purity of fibroblast cultures was confirmed by staining for vimentin and stain resistance for cytokeratin, desmin, and S-100, using the indirect immunofluorescence procedure, as previously described [14]. All-trans retinoic acid (RA) treatment of cell cultures: RA was dissolved in dimethylsulfoxide (DMSO) to a concentration of 10 −2 M, and this solution was diluted into DMEM/F12 to different concentrations and stored at 4 °C for immediate use or frozen in aliquots at −20 °C. Medium containing 0.1% DMSO, which is the highest final concentration used in these experiments, served as the control.
The numbers of cells in the cultures incubated with RA were estimated using the cell counting Kit-8 (Dojin Laboratories, Kumamoto, Japan) containing 2-(2-methoxy-4nitrophenyl) −3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium (WST-8) and 1-methoxyphenazine methosulfate (1-methoxy-PMS). Briefly, HSFs were seeded into 96-well plates coated with rat tail tendon type I collagen (Sigma, St. Louis, MO) at an initial density of 10 5 cells/well and preincubated with DMEM/F12 without fetal bovine serum (FBS) for 24 h. Various concentrations of RA (from 10 −9 M to 10 −4 M) or control medium were added. The cultures were incubated for a further 24 or 48 h. The solution of WST-8 and 1-methoxy-PMS was then added to each well, and the plates were incubated for 1 h, after which the absorbance at 450 nm was measured to determine the amount of formazan dye generated by dehydrogenases in cells, which is directly proportional to the number of living cells.
To extract total RNA or total protein for real-time PCR and western blot analysis, HSFs were plated in standard plates coated with rat tail tendon type I collagen with DMEM/F12 in 10% FBS at the same cell density for 24 h. The medium was changed to DMEM/F12 without FBS for another 24 h. Finally, these cells were grown in medium containing RA (from 10 −9 M to 10 −5 M) or in medium containing 0.1% DMSO as an untreated control. After 24 h of culture, total RNA was extracted. In a second experiment, cells were grown in control medium or in medium with RA at concentrations of either 10 −7 M or 10 −6 M. Total RNA was extracted after 12 h, 24 h, or 48 h, and total protein was extracted after 24 h or 48 h. Quantitative real-time PCR analysis and western blot analysis were undertaken on extracted total RNA and homogenized total protein, respectively. After 48 h of culture, the morphology of fibroblasts was also observed with light microscopy in control medium and in medium with 10 −7 M RA.
Indirect immunofluorescence: Fibulin-1 was visualized using standard immunohistochemistry in guinea pig sclera from animals fed RA or PO, and in both control and RA-treated HSFs. Briefly, fixed sections or cells were washed three times with PBS, covered with 10% normal donkey serum diluted in PBS, and incubated for 20 min at 37 °C. The slides were incubated at 4 °C overnight with primary antibodies (antifibulin-1 diluted to 1:50 in PBS, anti-aggrecan diluted to 1:50 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA). Cells and sections were incubated in PBS without primary antibodies as a negative control. The antibody-treated and negative control sample slides were washed with PBS and exposed to DyLight 488-conjugated anti-rabbit immunoglobulin G (IgG) antibodies or Dylight 594conjugated anti-goat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:1,000 in PBS at 37 °C for 60 min. The slides were washed in PBS three times, and cell nuclei were stained with Hoechst 33342 or propidium iodide (Sigma). Immunofluorescence images were taken using a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany).
Quantitative real-time PCR analysis: Real-time PCR analysis was used to show the expression of fibulin-1 at the transcription level. Total RNA was extracted from cultured HSFs using Trizol Reagent (Invitrogen). Complemenary DNAs (cDNAs) were synthesized with 4 μg of total RNA, 0.4 μl random primer, 0.5 μl dNTPs, and 200 U MMLV reverse transcriptase, 5× RT buffer (4 μl) at 37 °C for 1 h, followed by 95 °C for 3 min, using a TaqMan Reverse-Transcription kit from Promega (Promega, Madison, WI). An aliquot of the resulting single-stranded cDNA was used in the PCR experiments. Based on the sequences reported in the GenBank database (Table 1), primers were designed for Fbln1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with Primer Express Software (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed with an ABI7500 Fast Real-Time PCR System (Applied Biosystems , Foster City, CA). A typical reaction was performed in 50 μl, consisting of 5 μl of cDNA and 10 μl of 5× SYBR Green I PCR buffer, containing the specific primer pairs (final 10 pmol each). The PCR temperature cycle was 93 °C for 3 min, followed by 40 cycles at 93 °C for 30 s, 55 °C for 45 s, and 72 °C for 45 s, with SYBR Green fluorescence recorded at the end of each elongation segment. The PCR reaction was followed by a melting curve analysis: 93 °C for 10 s, 55 °C for 10 s, linear increase to 93 °C at 0.1 °C/s, with continuous SYBR Green fluorescence recording. The primer pairs used in this study for Fbln1 and GAPDH were validated as follows: they gave a single PCR product, as verified by melting curve analysis, agarose gel electrophoresis, and DNA sequencing; and the distribution of the PCR sigmoids was linear (r was 0.99 to 1) over 5 log units of template concentration with an efficiency of 1.85-1.98. The critical cycle of each sigmoid PCR curve was calculated by the ABI 7500 Fast Real-Time PCR System as the PCR cycle corresponding to the maximum of the second derivative. Total cDNA copy number from each cell culture sample was analyzed by the 7500 Fast Real-Time PCR Systems for Fbln1 and GAPDH. All copy numbers from each sample were compared to the GAPDH cDNA copy number from corresponding samples. Western blot analysis: Western blot analysis was used to show the expression of fibulin-1 at the protein level. Briefly, cells were washed with PBS and lysed in ice-cold lysis buffer (Shanghai Xinghan Sci & Tech Co. Ltd, Shanghai, China). After cell debris was removed by centrifugation at 15,000g at 4 °C for 30 min, the protein concentration was detected by BAC kits (Shenneng Bocai Biotechnology Co. Ltd, Shanghai, China). Protein (40 μg) was loaded in each lane of 10% sodium dodecyl sulfate-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes for electrophoresis, and blocked in Tris Buffered Saline with Tween (TBST) (5% fatfree dry milk, 0.1% Tween-20, 150 mM NaCl, and 50 mM Tris at pH 7.5) for 1 h. The membranes were exposed to 1:500 anti-fibulin-1 antibodies and incubated overnight at 4 °C. The same blots were then stripped and reanalyzed using anti-GAPDH antibodies (Beijing Biosynthesis Biotechnology Co. Ltd., Beijing, China) as an internal control. The membranes were then incubated with a secondary horseradish peroxidase (HRP)-labeled antibody (Beijing Biosynthesis Biotechnology Co. Ltd.) for 1 h. Protein bands were visualized with the use of a chemiluminescence Phototope (R)-HRP Western Blot detection system (Cell Signaling Technology, Inc., Danvers, MA) and exposed to a negative film, developed, and fixed. The film was scanned and analyzed with Quantity One Analyzer Software (Bio-Rad Laboratories, Santa Cruz, CA). The relative level of protein expression was expressed as the density ratio of the protein compared to GAPDH in the same sample. Independent experiments were performed and repeated three times.

Effects of gavage with retinoic acid on ocular size and refraction:
Ocular size was increased by treatment with RA with a difference in axial length (axial distance from the cornea to the retina) of 97.5 µm (RA, 7.743±0.005 mm; PO, 7.628±0.009 mm, p<0.01), with most of the elongation due to an increase in the vitreous chamber depth (difference of 101.8 µm). For animals of this age, a 50-µm increase in ocular length is equal to approximately a −2D refractive shift [15]. We found here that RA treatment caused a myopic shift in refractive error of −4.8D (RA, 1.73±0.8D; PO, +6.5±1.6D, p<0.01).

Expression of fibulin-1 and aggrecan in guinea pig sclera:
Fibulin-1 and aggrecan immunohistochemical staining were observed in scleras from RA-or PO-treated guinea pigs. In all eyes, fibulin-1 was localized in scleral extracellular matrices in parallel with the distribution of aggrecan. For the PO-fed guinea pigs, aggrecan was most strongly labeled in posterior sclera and was irregularly arranged between collagen fibrils and collagenous lamellae, with scleral fibroblasts located between them. In PO eyes, fibulin-1 immunoreactivity was  low and was detectable in and around fibroblasts ( Figure 1E-H). In RA-fed guinea pigs, the expression of fibulin-1 in the sclera was substantially increased, whereas the expression of aggrecan was depressed ( Figure 1I-L).  where density values were compared to GAPDH density. The asterisk indicates a significant difference relative to the control (p<0.05).  Figure 3A). Concentrations of RA that reduced cell numbers were less effective in the upregulation of Fbln1 mRNA. To find the effective time that RA took to upregulate Fbln1 mRNA expression in HSFs, total RNA prepared from cells treated with 10 −7 M RA for different times (12 h, 24 h, 48 h) was analyzed and compared with total RNA from control cultures ( Figure 3B). RA at 10 −7 M induced the most marked expression of Fbln1 mRNA in HSFs, and the effect was time dependent. There were no significant changes in Fbln1 mRNA in HSFs after incubation with RA for 12 h, but Fbln1 mRNA levels were significantly increased after treatment of HSFs with 10 −7 M RA for 24 h and 48 h (p<0.001 in both cases), with the latter showing a dramatic increase of 9.4 times the control ( Figure 3B). Retinoic acid-induced changes in fibulin-1 protein levels in cultured human scleral fibroblasts: Protein prepared from the cells treated with RA concentrations of 10 −7 or 10 −6 M for either 24 h or 48 h were analyzed and compared with controls (medium with 0.1% DMSO). The relative protein levels of fibulin-1 in HSFs incubated with RA are represented in Figure  4. RA upregulated the fibulin-1 protein level in a timedependent manner. The fibulin-1 protein expression was significantly increased after the cells were treated with RA at 10 −7 M at both 24 h and 48 h (p<0.05). Morphological changes in human scleral fibroblasts and fibulin-1 immunohistochemical staining after retinoic acid treatment: HSFs seeded onto collagen I and cultured in 10 −7 M RA or medium with 0.1% DMSO (control) for 48 h showed morphological changes. Under the inverted phase contrast microscope, control HSFs displayed many spreading protrusions on collagen-coated standard plates ( Figure 5A). After RA treatment HSFs became smaller with little evidence of any protrusions ( Figure 5B). With fibulin-1 immunohistchemistry, fibulin-1 was found to be weakly expressed in the cytoplasm of control cells with a uniform distribution ( Figure 6D-F). In cells cultured in a medium containing 10 −7 M RA for 48 h, fibulin-1 expression was significantly increased ( Figure 6G-I).

DISCUSSION
Fibulin-1, which is an important ECM molecule, is associated with tissue remodeling in many systems [1,2]. Recent work has shown that fibulin-1 is expressed in human sclera [7]. Here, we provide evidence that fibulin-1 protein is expressed in vivo in guinea pig sclera and that gavage with RA leads to increased expression of fibulin-1 and decreased expression of aggrecan. We further show that fibulin-1 is expressed in HSFs grown in culture, where expression is also regulated by RA. These results suggest that RA regulates the expression of fibulin-1 similarly in vivo and in vitro and that the molecular pathways involved may therefore be further studied using cultured fibroblasts.
RA can affect scleral ECM during scleral remodeling [10]. RA appears to regulate the expression of aggrecan in skeletal systems and affect cartilage matrix homeostasis [11]. However, the effect of RA on aggrecan and fibulin-1 in the sclera has not been previously reported. In this study we fed RA to normal guinea pigs, and the eyes rapidly elongated and became myopic. Using immunohistochemical staining, we found that fibulin-1 and aggrecan were distributed in a similar fashion in normal guinea pig sclera, but fibulin-1 staining was weak, whereas aggrecan staining was stronger. In the sclera from RA-fed animals, the expression of fibulin-1 was increased, while there was a loss of scleral aggrecan staining. Similar results have been reported on cultured cartilage tissue where exposure to RA led to the extensive loss of aggrecan from the tissue [16]. The reciprocal relationship between fibulin and aggrecan observed in our study may be due to the ability of fibulin-1 to enhance the cleavage of aggrecan by a disintegrin-like and metalloprotease with thromobospondin type-1 [6,17]. This suggests that the pathway leading to increased eye growth may involve upregulation of fibulin-1 by RA and degradation of aggrecan.
The expression of fibulin-1 was induced by RA in a dosedependent manner in cultured HSFs, as shown by both realtime PCR and western blot analysis. A 10 −7 M concentration of RA maximally increased the expression of fibulin-1 protein in HSFs. This concentration is similar to the concentration of RA shown to inhibit proteoglycan synthesis in cultured chick sclera [10], and we have shown here that this concentration does not reduce the number of cells that grow under cultured conditions. Since RA appears to be an important molecular signal in the control of eye size [9,10,[18][19][20], it is tempting to relate the capacity of RA to inhibit aggrecan synthesis in cultured sclera to its effect on eye growth.
Although the functions of fibulin-1 in the sclera remain unclear, our finding of fibulin-1 expression induced by RA, which is a molecule known to be involved in the regulation of eye growth, suggests that it is worth looking at the role of fibulin-1 in scleral remodeling. Interactions between fibulin-1 and aggrecan may be important, and the modulation of aggrecan levels and distribution could play a key role in changing the scleral creep rate [5], which is associated with axial elongation of the eye and the development of myopia [21]. Further research on the function of fibulin-1 and its interaction with aggrecan in sclera during the development of myopia is warranted.